Ramakrishnan S, Sharma H W, Farris A D, Kaufman K M, Harley J B, Collins K, Pruijn G J, van Venrooij W J, Martin M L, Narayanan R
Department of Oncology, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110, USA.
Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10075-9. doi: 10.1073/pnas.94.19.10075.
Telomerase, a ribonucleoprotein complex, adds hexameric repeats called "telomeres" to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.
端粒酶是一种核糖核蛋白复合体，它将被称为“端粒”的六聚体重复序列添加到染色体DNA不断增长的末端。由于其低表达水平，哺乳动物端粒酶的特性一直难以捉摸。我们描述了一种生物信息学方法来富集和表征人类端粒酶复合体。使用局部序列同源性搜索方法，我们检测到端粒酶的嗜热四膜虫p80亚基与自身抗原Ro60的相似性。抗Ro60抗体免疫沉淀了端粒酶活性。Ro60和p80蛋白可被针对任一蛋白的抗体交叉识别。端粒酶活性和端粒酶复合体的RNA组分在来自Ro60抗体沉淀材料的天然凝胶中定位于一个双峰。富集的材料在体外以RNA模板依赖的方式与TTA GGG探针特异性结合。针对该双峰的多克隆抗体也免疫沉淀了端粒酶活性。这些结果表明端粒酶蛋白存在进化保守性。
